1. Field of the Invention
The present invention relates to photodynamic therapy and chemotherapy, wherein a xe2x80x9cparachutexe2x80x9d structure is used to deliver a therapeutic agent to a defined subcellular location, and therefore the action of the therapeutic agent, such as destroying tumor cells, is achieved more effectively.
2. Invention Disclosure Statement
During the last two decades, there has been an increasing interest in utilizing photosensitizers for cancer therapy, where the technique is known as Photodynamic Therapy (PDT). Dougherty T J, Photosensitizers: Therapy and detection malignant tumors, Photochem. Photobiol, 45(6): 879-889, (1987) and Dougherty T J, Photodynamic therapy, Photochem. Photobiol, 58(6): 895-900, (1993) describe a technique where singlet oxygen, oxygen radicals, and superoxides/peroxides are produced by in situ photosensitization of previously applied chromophores, and thus destroy the malignant cells. The technique utilizes non-toxic, photosensitizing drugs in combination with non-hazardous irradiation, and has the potential of being more selective yet no less effective when compared with the commonly used chemotherapy or radiotherapy. It is therefore expected to increase the quality of life for treated patients Moreover, Sokolov V. et al., Multicourse PDT of malignant tumors: the influence of the primary tumor, metastatic spreading and homoestasis in cancer patients. SPIF Biomedical Optics 3191, 322-329 (1996) shows that no scars are observed after removal of the tumors using this technique and the surrounding muscular structures are fully functional.
The photosensitizers used in PDT need to have a high quantum yield for singlet oxygen production, as well as characteristics of high affinity and selectivity for the malignant tissue. Porphyrins have a high quantum yield for the formation of an excited triplet state. The difference between the energies of triplet state and ground-state oxygen makes them good energy donors to transfer its energy to the ground state to form singlet oxygen. One experimental drug known as Photofrin II (a purified version of hematoporphyrin derivative) is currently involved in randomized clinical trials. Other photosensitizing drugs used in photodynamic therapy procedures include phthalocyanines, merocyanine 540, substituted purines, xanthenes (Rhodamine 123), cationic cyanine dyes, chlorines, chalcogenapyrylium dyes containing selenium or tellurium atoms in the chromophore, phenothiazinium derivatives, benzophenoxoniums (Nile Blue A) and triarylmethanes (Methylene Blue, Victoria Blue BO [VB-BO]).
Illumination of the target site by an appropriate light source, such as a sunlamp, an argon-pumped dye laser, or more recently, diode lasers, induces the cytotoxic effect on the cells of the target site by one of two proposed mechanisms. In Type I photosensitization, the electronically excited drug reacts directly with a biological substrate, forming radicals which can initiate subsequent radical reactions that induce cytotoxic damage. Type II photosensitization involves energy transfer from the electronically excited drugs to oxygen, producing singlet oxygen which subsequently produces cytotoxic oxygenated products destroying the cell membrane of tumor (and vascular endothelial) cells directly.
Although remarkable results have been obtained in some PDT trials, several problems remain. The low solubility of some of the sensitizers reduces their usefulness for intravascular administration because it can provoke thromboembolic events. The use of liposomes as transport vehicles can overcome the problem of the solubility, but there still remains the difficulty of directing the sensitizers to specific target sites. Moreover, liposomes administered intravenously to subjects are rapidly accumulated in the reticuloendothelial system often inducing several severe allergic reactions. High liposome concentration is thereby rapidly achieved in organs with fenestrated capillaries, such as the liver, spleen, and bone marrow. Liposomal systems can be effective in treating tumors that infiltrate these organs (such as hematologic malignancies), but have been less useful in treating targeted tumors in other anatomical locations.
Very high systemic doses of the sensitizer must often be given to achieve therapeutic levels at irradiated tumor sites, hence many sites in the body are nonselectively infiltrated by the sensitizer. As the amount of the applied photosensitizer increases, the chances of its accumulation in normal tissues and the accompanying risk of damaging non-malignant sites profoundly increases.
There have been many attempts to increase the specificity of the photosensitizers for the tumor and thereby concentrating a sufficient amount of molecules in the tumor cells. Moser J G xe2x80x9cDefinition and general properties of 2nd and 3rd generation photosensitizersxe2x80x9d and xe2x80x9ccarrier and delivery systemsxe2x80x9d in Photodynamic tumor therapy 2nd and 3rd generation photosensitizers, pp. 3-8 and 127-136 respectively, [Moser, J G ed, Harwood Academic Publishers, London, (1998)] describe an approach of the coupling of several molecules of the dye with tumor specific antibodies which resulted in excellent phototoxicity in vitro. However, in vivo this method failed due to the recognition of the vast complex by the reticuloendothelial system, subsequent accumulation in the liver, and digestion of the compound before it reached the target site. Hirth A et al., New biotinylated phthalocyanines for the photodynamic therapy of cancer. SPIE Biomed. Optics 3191, 309-314, (1997) disclosed a more promising approach using polyphasic tumor targeting. Here, the antibodies are coupled with only minimum deviation substituents like biotin, so that they are not recognized by the reticuloendothelial system. After clearing the excessive biotinylated antibodies in the system, the photosensitizer coupled to avidin can be administered and accumulate at the cells labeled by the antibody. This method still has to be developed further for in vivo use in tumor therapy. But even if the targeting of the dye to the tumor would be successful, still an amount of at least 107 photosensitizer molecules per cell has to be reached for the effective destruction of the tumor, which none of the described techniques have successfully achieved.
The reduction of the amount of photosensitizer molecules necessary for successful therapy could be achieved by more selective targeting of the dyes to the sites in the cell, where the compounds can develop the most effective action for the destruction of the malignant cells. Specific subcellular targeting of photosensitizers was only rarely performed and observed. One example is Rhodamine 123 targeting mitochondrial membranes. Matz et al, Fluorescent proteins from nonbioluminescent Anthozoa species, Nat. Biotechnol, 17(10): 969-73, (1999) describes red-fluorescent targeting proteins for achieving the same effect. Kessel D, Use of fluorescent probes for characterizing sites of photodamage, The Spectrum 6(2), 1-6, (1993) describes other examples and techniques of detection. The frequent localization of bacteriochlorophyll derivatives is at the Golgi apparatus. However, both localizations are not very deleterious for cancer cells since these cells obtain their energy from basal cytoplasmic metabolism.
In summary, there are various attempts to increase the specificity of phototherapeutic drugs and thereby to reduce the doses necessary for the successful photodynamic therapy of tumors. However, no satisfying solution to this problem has been presented so far. The present invention provides a novel method to enhance the effect of photosensitizers or other therapeutic compounds on cells to achieve successful therapy with reduced doses of the drugs.
It is an object of the present invention to provide a system for and method of localizing therapeutic compounds at the most sensitive sites in a cell, thereby reducing therapeutic doses of the drug and in turn reducing the side effects of the therapy.
It is another object of the present invention to localize therapeutic compounds at cell membranes which results in a reduction of the dosage needed by a factor of 10-100 or more while achieving the same therapeutic effect.
It is a further object of the present invention to localize therapeutic compounds not only at a cell membrane, but also at defined distances to the membrane within the cells to optimize the destroying properties of the respective drug.
Still another object of the present invention is to modify the hydrophilic residues of the delivery structure so that it can also carry signals for targeting of the therapeutic complex to a specific tissue type.
Briefly stated, the present invention provides a drug delivery system wherein a xe2x80x9cparachutexe2x80x9d structure is coupled to a therapeutic compound. The xe2x80x9cparachutexe2x80x9d structure comprises hydrophilic branched molecules with a defined action diameter, where action diameter is the distance between the hydrophilic moieties that is necessary to localize the complex in the cell membrane. The complex (a parachute structure coupled with a therapeutic compound) is either fixed at a cell membrane or delivered to a defined distance from the membrane within the cell. The membrane-anchoring/localizing effect of the parachute is achieved by hydrophilic structures linked with a branching unit of desired therapeutic compounds (such as photosensitizers and chemotherapeutics). For example, di-glucosamine, a hydrophilic structure of a defined action diameter, functions to retard the molecules at the cell membrane, and thus avoids or slows deeper penetration of the therapeutic compounds into the cell during the short time span before irradiation. The defined action diameter can be achieved by using a branching unit, which can be triazine trichloride or trimesinic acid trichloride, to connect the therapeutic compound such as porphyrin over a diamide spacer bridge with two glucosamine residues. Furthermore, the parachute structures can be connected by a spacer (e.g. xcex2-amino acids, xcex3-amino butyric acid, poly-amino acids, aliphatic, aromatic or heterocyclic molecules) instead of directly binding to the therapeutic compound, so that the therapeutic compounds can be localized within the cells at a defined distance from the cell membrane. A spacer containing a breaking point (for example, it can be cleaved by the action of cellular enzymes) can determine the time span, during which the drug exhibits its therapeutic activity. The hydrophilic residues can also carry signals for targeting the parachute-therapeutic complex to a defined tissue type. This can be mediated by an antibody which is specific for a tumor marker. Alternatively, a Biotin can be attached at C6 position of the sugar and then react with an Avidin-labeled tumor-specific antibody. The Biotin-Avidin system has the advantage of minimizing the size of the delivery structure so that it will not be recognized by the reticuloendothelial system and get destroyed. The parachute function may also be achieved by other, more bulky hydrophilic structures such as oligosaccharides connected to the branching unit. Such sugar oligomers have specific attachment points to cell selecting, and therefore do not need additional molecular structures to target a specific tumor tissue. The use of the parachute structure gives the advantages of being able to localize a photosensitizer or chemotherapeutic drug at the site within a cell where it can destroy the tumor cell most effectively. This reduces the level of necessary systemic doses of the drugs, promotes drug excretion, and therefore considerably reduces side effects of the therapy.
The above, and other objects, features and advantages of the present invention will become apparent from the following description read in conjunction with the accompanying drawings.